Creating a TALE protein with unbiased 5′-T binding

Tsuji, Shogo; Futaki, Shiroh; Imanishi, Masahito

doi:10.1016/j.bbrc.2013.10.060

Cited by 21 publications

(19 citation statements)

References 23 publications

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“…This hypothesis is supported by (i) the natural TALE TalC, which contains a tryptophan to cysteine substitution in repeat −1 and a first canonical repeat with the RVD NS1 and by (ii) the functionality of W232C and W232A mutations in AvrBs3 if the repeat region starts with NS1. Our data provide additional explanations to previous studies, in which W232 of TALEs was mutated (17,25). Notably, we confirmed data obtained by Tsuji et al.…”

Section: Discussionsupporting

confidence: 82%

“…Notably, we confirmed data obtained by Tsuji et al. (25) for a dTALE that starts with HD1 and contains 14.5 repeats. In contrast, Doyle et al.…”

Section: Discussionmentioning

confidence: 99%

“…In the latter case, however, the results were reported to be highly variable (17). Recently, T 0 -independent NTRs were generated by mutation of the ‘RVD-loop’ of repeat −1 (22,25). Notably, a G 0 -specific NTR was generated by the double amino acid substitution W232R/Q231S in the dTALE Avr15 (14.5 repeats; NI1; optimal EBE) (22).…”

Section: Discussionmentioning

confidence: 99%

“…In our study, TALEs, containing HD1 always worked less well if combined with an EBE that starts with G 0 . Interestingly, A 0 -specific and C 0 -specific but not G 0 -specific NTRs combined with a repeat region that starts with HD1 were described (25). This might be due to the fact that G 0 in EBEs of TALEs is unfavored if the first RVD of the canonical repeats is HD1 (as seen with AvrBs3 and the ARTrep18 constructs).…”

Section: Discussionmentioning

confidence: 99%

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Repeat 1 of TAL effectors affects target specificity for the base at position zero

Schreiber

Bonas

2014

Nucleic Acids Research

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AvrBs3, the founding member of the Xanthomonas transcription-activator-like effectors (TALEs), is translocated into the plant cell where it localizes to the nucleus and acts as transcription factor. The DNA-binding domain of AvrBs3 consists of 17.5 nearly-identical 34 amino acid-repeats. Each repeat specifies binding to one base in the target DNA via amino acid residues 12 and 13 termed repeat variable diresidue (RVD). Natural target sequences of TALEs are generally preceded by a thymine (T0), which is coordinated by a tryptophan residue (W232) in a degenerated repeat upstream of the canonical repeats. To investigate the necessity of T0 and the conserved tryptophan for AvrBs3-mediated gene activation we tested TALE mutant derivatives on target sequences preceded by all possible four bases. In addition, we performed domain swaps with TalC from a rice pathogenic Xanthomonas because TalC lacks the tryptophan residue, and the TalC target sequence is preceded by cytosine. We show that T0 works best and that T0 specificity depends on the repeat number and overall RVD-composition. T0 and W232 appear to be particularly important if the RVD of the first repeat is HD (‘rep1 effect’). Our findings provide novel insights into the mechanism of T0 recognition by TALE proteins and are important for TALE-based biotechnological applications.

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Section: Discussionsupporting

confidence: 82%

“…Notably, we confirmed data obtained by Tsuji et al. (25) for a dTALE that starts with HD1 and contains 14.5 repeats. In contrast, Doyle et al.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Repeat 1 of TAL effectors affects target specificity for the base at position zero

Schreiber

Bonas

2014

Nucleic Acids Research

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“…The number and position of cryptic N- and C-terminal repeats are typically inflexible, though alternative repeat −1 modules have recently been described (24,25). We tested acBat1 deletion derivatives to test if this paradigm applies to Bat1.…”

Section: Resultsmentioning

confidence: 99%

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

Lange

Wolf

Dietze

et al. 2014

Nucleic Acids Research

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The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution.

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TAL Effector DNA-Binding Principles and Specificity

Richter

Streubel

Boch

2016

TALENs

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Transcription activator-like effectors (TALEs) are proteins with a unique DNA-binding domain that confers both a predictable and programmable specificity. The DNA-binding domain consists typically of 34-amino acid near-identical repeats. The repeats form a right-handed superhelical structure that wraps around the DNA double helix and exposes the variable amino acids at position 13 of each repeat to the sense strand DNA bases. Each repeat binds one base in a highly specific, non-overlapping, and comma-free fashion. Although TALE specificities are encoded in a simple way, sophisticated rules can be taken into account to build highly efficient DNA-binding modules for biotechnological use.

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Creating a TALE protein with unbiased 5′-T binding

Cited by 21 publications

References 23 publications

Repeat 1 of TAL effectors affects target specificity for the base at position zero

Repeat 1 of TAL effectors affects target specificity for the base at position zero

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain

TAL Effector DNA-Binding Principles and Specificity

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